Pharmacologic remedy of many brain diseases is difficult because of the powerful drug exclusion properties of the blood-brain barrier (BBB). Chemical isolation of the vertebrate brain is achieved through the highly integrated, anatomically compact and functionally overlapping chemical isolation processes of the BBB. These include functions that need to be coordinated between tight diffusion junctions and unidirectionally acting xenobiotic transporters. Understanding of many of these processes has been hampered, because they have been experimentally difficult and expensive to disentangle in intact rodent models. Consequently, many of the processes are not well mimicked by in vitro or ex vivo BBB models.
In drug research it is extremely important to determine brain penetration both for drug candidates with CNS therapeutic potential but also for compounds which can cause CNS mediated side effects. There are in principle two strategies to measure brain penetration, a) determination of the rate of brain uptake at the initial state and b) the extent of brain exposure at the static state. The former is regarded as the most relevant because it is not compromised by metabolism, plasma binding or non specific brain binding due to the short exposure time. Because of their importance numerous methods have been developed to evaluate the rate of brain penetration. In situ brain perfusion, cell-based MDR1-MDCK assay and the PAMPA method are the most common assays in the pharmacological industry to determine BBB permeability. In situ brain perfusion is considered a golden standard method for measuring BBB permeability but in the pharmaceutical industry it does not fulfil the requirements of a method with high throughput and short-term feed back during the earliest step of drug discovery due to its labour intensity and high cost per tested candidate. For this reason the industry tend to use the high throughput but inaccurate in vitro models to assess BBB penetration.
In general, the in vitro, based, assays are regularly and routinely applied in the pharmaceutical early drug discovery phases and despite that there are major limitations by these assays most pharmaceutical companies use large batteries of in vitro screens. However, testing compounds in a large number of in vitro assays may not always reflect the in vivo behaviour. In fact, it is not unusual that compounds that have acceptable in vitro profiles turn out to have inadequate in vivo profiles. On the contrary, compounds may be discarded for wrong reasons.
Hence, there is a demand for intermediate models that are more reliable than in vitro models and at the same time faster and cheaper than traditional vertebrate in vivo models.
The vertebrate blood-brain barrier (BBB) represents the physiologic barrier between the brain tissue and blood vessels, which restricts the exchange of solutes and regulates absorption of exogenic agents (e.g. drugs) from the blood into the brain. The function of the central nervous system (CNS) requires a highly regulated extra-cellular environment. Anatomically the BBB in vertebrates is comprised of microvascular endothelia cells interconnected via highly specialized tight junctions (TJs), which provide a diffusion barrier and thus play a central role for permeability. Recently identified components of TJs include the claudins, a family of four-transmembrane-span proteins that are suggested to be responsible for the barrier-function of TJs (Turksen and Troy 2004). Penetration of BBB is one of the major hurdles in the development of successful CNS drugs. On the other hand, when penetration of the BBB occurs it may cause unwanted side effects for peripheral acting drugs (Schinkel 1999) (for review see Pardridge 2002).
BBB penetration is usually classified as chemistry- or biology-based. The chemistry-based penetration is linked to the lipid mediated passive diffusion, which depends on physiochemical properties of the molecule, i.e. small hydrophobic molecules tend to penetrate the BBB more readily than large and hydrophilic molecules. The biology-based penetration involves compounds that are substrates for the endogenous BBB influx or efflux transport systems, e.g. many small molecules (e.g. drugs) have shown to be substrates for the P-glycoprotein (P-gp) transporter. The P-gp's are transporter proteins located in the walls of the cells that make up the BBB (Schinkel 1999) and they are conserved among taxa as diverse as protozoa, plants, insects and mammals (Gaertner et. al. 1998). P-gp's are present in many cell-types and they play important roles in drug absorption, disposition, metabolism, and toxicity (Xia et al. 2006).
Obviously, it is crucial to have an understanding of the BBB penetration in drug discovery projects and preferably, this should be obtained without using excessive number of in vivo studies. Consequently, several in vitro absorption models are developed to predict the in vivo behaviour of test compounds. However, even complex in vitro models which include the P-gp transporter systems (Di and Kerns 2003, Summerfield et al. 2005) seem not to meet the intricate complexity of the BBB and therefore may not describe the in vivo behavior very well. The popular CaCo-2 model, developed to predict oral uptake, showed to be less useful for predicting brain penetration and the MDR1-MDCK assay, which is widely applied in industry, is mainly used to diagnose a Pgp efflux transport and recent studies have confirmed the low predictability of passive BBB permeability of this model (Summerfield et al., 2007). In an extensive BBB absorption study 22 compounds were tested in ten different in vitro BBB absorption models (Garberg 2005). None of the ten models showed correlation between in vitro and in vivo permeability. This indicates that specific BBB models not necessarily provide better prediction than non-BBB derived models. Furthermore, it was suggested that protein binding, blood-flow, metabolic stability and lipophilicity, as well as affinity for other transporters in the BBB are factors needed to be considered when predictions of in vivo brain distribution is to be made. Consequently, it seems as in vitro models are mainly suited for qualitative measurements of compounds that penetrates BBB by passive diffusion or compounds that undergo efflux via the P-gp transporter (Garberg 2005).
In vertebrates, a physically separate blood-brain barrier (BBB), primarily engineered into the single-cell layer vascular endothelium, provides an obstacle to chemical attack. At this interface, strong selective pressures have produced the integration of at least two very different cell biologic mechanisms to prevent free movement of small molecules between the humoral and CNS interstitial compartments. (Abbott, 2005; Daneman and Barres, 2005; Neuwelt et al., 2008; Zlokovic, 2008). BBB vascular endothelium cells impede the traffic of drugs by virtue of specialized lateral junction components, including tight junctions, and asymmetrically arrayed ATP binding cassette (ABC) transporters. Tight junctions prevent paracellular diffusion of charged molecules, and asymmetrically arrayed transporters actively expel lipophilic molecules back into the humoral space (Löscher and Potschka, 2005). Together, these complimentary systems prevent the majority of xenobiotics from acting on vertebrate nervous tissue (Pardridge, 2005). Although in vivo and in vitro BBB models have confirmed the importance of these two components (Schinkel et al., 1997; Nitta et al., 2003), substantial limitations hinder progress (Garberg et al., 2005). A powerful BBB model system should combine molecular genetic, genomic, chemical biology, and integrative physiology tools to probe CNS-specific chemoprotective physiology.
Recent research have shown that insects possess neural barriers that differs anatomically from the vertebrate barriers but also possess a number of highly important and relevant structures that is shared with the vertebrate barrier making the insect brain barrier a superior candidate for BBB permeability studies. Thus it has been shown that the insect barrier cells (glia) contain pleated septate and tight junctions nearly identical to the proteins that make up the vertebrate tight junctions (Wu and Beitel, 2004; Pardridge, 2005). Furthermore, it has been shown that insects possess a homology to the major ATP binding cassette (ABC) transporter (MDR/Pgp). It has also been shown that the active transporter homolog is localised at the hemolymph barrier, indicating that the subperineural glia in insects, like the vascular endothelium in vertebrates, possesses both tight junction barriers and active efflux transporters. These conclusions strengthen the utility of the insect BBB as a relevant model for screening and documentation of brain penetration in drug research. In further support of the functional relevance of the insect brain barrier model it has been shown in Drosophila that manipulation of the two principal barrier mechanisms by using measures relevant for the vertebrate barriers will open up both the diffusion and the transport barriers in Drosophila. Thus treatment of Drosophila with the known vertebrate MDR1/Pgp transport blocker Cyclosporin A (CsA) increased the ABC substrate in the Drosophila brain (Mayer et al., 2009) and osmotic manipulation of the grasshopper (L. migratoria) open up the diffusion barrier at similar concentration as is used in correspondent vertebrate studies (Andersson et al., 2009). Thus the coincident localization of the diffusion and the xenobiotic transport barriers demonstrate that insects combine vertebrate-like drug exclusion mechanisms to maintain a chemical barrier to the brain. These observations are the base for an ex vivo insect model with high utility in the early screening and documentation of candidate compounds in early drug discovery phase. In contrast to other models (e.g. in vitro models including the PAMPA model) our model is characterised by the properties that are the bases for an appropriate function of a brain barrier and in this sense highly relevant for prediction of brain penetration in vertebrates including human.
In CNS drug discovery there is a need for efficient screening of compounds aimed at targets within the CNS system. This screening is preferentially performed in insect models with intact BBB function and will contribute to a positive selection of compounds penetrating the BBB. Such screening comprises low molecular weight compounds within a number of indications (e.g. pain, epilepsy, Parkinson, schizophrenia, Alzheimer, sleep disorders, anxiety, depression, eating disorders, drug abuse including smoking).